The Lipid Composition and Physical Properties of the Yeast Vacuole Affect the Hemifusion–Fusion Transition

Abstract

Yeast vacuole fusion requires the formation of SNARE bundles between membranes. Although the function of vacuolar SNAREs is controlled in part by regulatory lipids, the exact role of the membrane in regulating fusion remains unclear. Because SNAREs are membrane-anchored and transmit the force required for fusion to the bilayer, we hypothesized that the lipid composition and curvature of the membrane aid in controlling fusion. Here, we examined the effect of altering membrane fluidity and curvature on the functionality of fusion-incompetent SNARE mutants that are thought to generate insufficient force to trigger the hemifusion-fusion transition. The hemifusion-fusion transition was inhibited by disrupting the 3Q:1R stoichiometry of SNARE bundles with the mutant SNARE Vam7pQ283R. Similarly, replacing the transmembrane domain of the syntaxin homolog Vam3p with a lipid anchor allowed hemifusion, but not content mixing. Hemifusion-stalled reactions containing either of the SNARE mutants were stimulated to fuse with chlorpromazine, an amphipathic molecule that alters membrane fluidity and curvature. The activity of mutant SNAREs was also rescued by the overexpression of SNAREs, thus multiplying the force transferred to the membrane. Thus, we conclude that either increasing membrane fluidity, or multiplying SNARE-generated energy restored the fusogenicity of mutant SNAREs that are stalled at hemifusion. We also found that regulatory lipids differentially modulated the complex formation of wild-type SNAREs. Together, these data indicate that the physical properties and the lipid composition of the membrane affect the function of SNAREs in promoting the hemifusion-fusion transition.

The Yeast ATP-binding Cassette (ABC) Transporter Ycf1p Enhances the Recruitment of the Soluble SNARE Vam7p to Vacuoles for Efficient Membrane Fusion

“…PI3P is generated during fusion (33) by the phosphatidylinositol kinase Vps34p (9), and the absence or sequestering of PI3P has severe negative effects on the fusion machinery. This lipid is required for the recruitment of Vam7p as well as the formation of membrane raft domains and the interactions between SNAREs and HOPS (3,44,45). PI3P levels can be reduced through the phosphatase activity of Ymr1p or the kinase activity of Fab1p (46,47), although it remains unknown whether either Ymr1p or Fab1p functions during vacuole homotypic fusion.…”

Class C ABC transporters andSaccharomyces cerevisiaevacuole fusion

“…58 Vacuole fusion can be augmented by multiple means including deleting the type 1 casein kinase Yck3, 59 altering osmolarity, 60 and increasing the number of SNAREs per vacuole. 17 Vacuoles purified from ybt1D yeast contained wild type levels of SNAREs suggesting that the augmented fusion was not caused by increased copies of SNAREs. It remains unclear whether deleting YBT1 affects Yck3 function or osmoregulation.…”

“…Fusion can occur through a hemifusion intermediate at the vertex ring where the outer leaflets of docked vacuoles fuse while the inner leaflets remain intact. [14][15][16][17] Thus, the positive curvature of vertex domains during docking changes to negatively curved domains upon hemifusion that contributes to destabilization of the bilayers. 18,19 Ultimately, the inner leaflets fuse and luminal contents mix.…”

“…However, it should be noted that adding recombinant Vam7 to in vitro fusion reactions can bypass blocks in fusion that occur prior to docking and trans-SNARE pairing. 10,17,90,91 Thus, it is possible that Ycf1 serves a second function during fusion that is linked to transport activity prior to docking. Because the in vitro fusion assay is devoid of toxic levels of Cd 2C and other soluble substrates, or glutathione S-transferase for that matter, we must suppose that Ycf1 carries out a novel ATPase dependent function.…”

The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain

“…This membrane microdomain is termed the vertex ring and is the site of fusion. Fusion can occur directly through the formation of a fusion pore or through an intermediate hemifusion state where the outer leaflets of membrane mix without fusing the inner leaflets, thus preventing content mixing (15)(16)(17)(18). Full fusion occurs when both leaflets fuse and luminal contents are mixed.…”